A central question in eukaryotic molecular biology is how specific DNA-binding proteins bind regulatory sequences to influence cell function and fate. The steroid/thyroid hormone receptors form a superfamily of ligand-dependent transcription factors that are believed to play a part in such cell function and fate. For example, it is known that these receptors transduce extracellular hormonal signal to target genes that contain specific enhancer sequences referred to as hormone-response elements (HREs). Each receptor contains a ligand-binding domain and a DNA-binding domain. The receptor undergoes a conformational change when it binds ligand which conformational change permits the receptor-ligand complex to bind its cognate response element and thereby regulate transcriptional activity of an associated promoter, which drives transcription of an operatively associated structural gene.
Sequence comparison and mutational analyses of hormone receptors such as glucocorticoid receptor (GR) have identified functional domains responsible for transcriptional activation and repression, nuclear localization, DNA binding, and hormone binding. The DNA binding domain, which is required in order to activate transcription, consists of 66-68 amino acids of which about 20 sites, including nine cysteines (C.sub.1 to C.sub.9), are invariant among different receptors. The modular structure of members of this receptor superfamily allows the exchange of one domain for another to create functional, chimeric receptors.
The hormone response elements are generally structurally related but in fact are functionally distinct. Those for GR (GRE), estrogen receptor (ERE), and thyroid hormone receptor response elements (TRE) have been characterized in detail; they consist of a palindromic pair of `half sites` (Evans, Science 240, 889 (1988); Green and Chambon, Trends In Genetics 4, 309 (1988)). With optimized pseudo- or consensus response elements, only two nucleotides per half site are different in GRE and ERE (Klock, et al., Nature 329, 734 (1987)). On the other hand, identical half sites can be seen in ERE and TRE, but their spacing is different (Glass, et al., Cell 54, 313 (1988)). Moreover, TRE has been shown to mediate transcriptional activation by transfected retinoic acid receptors (RARs) in CV-1 cells whereas non-transfected cells show no response. (Umesono et al., Nature 336, 262 (1988)). In other words, both TR and RR receptors can activate TREs.
It is, thus, surprising that the .beta.-retinoic acid response elements (.beta.RAREs) disclosed herein have a tandem repeat sequence as opposed to a palindromic sequence, and are much less susceptible to transcriptional activation by non-cognate receptors (e.g., estrogen receptor (ER), GR, thyroid hormone receptor (TR), etc.) than the known response elements (GRE, ERE, TRE). Also surprising is that constructs having .beta.RAREs in a wide variety of mammalian cells have shown robust retinoic acid (RA)-dependent induction in the absence of cotransfected retinoic acid receptor (RAR)-encoding expression vector. This discovery suggests that virtually all mammalian cells express a low level of endogenous .beta.RAR that is sufficient for efficient activation of vectors containing the .beta.RARE, but apparently below a threshold for activation of the previously studied TREs.
Thus, using transcriptional control regions comprising .beta.RARE and a functional promoter, it is now possible to provide recombinant DNA vectors containing a gene, the transcription (and, thereby, also expression) of which is under the control of a promoter, the transcriptional activity of which is responsive to (and increased by) retinoic acid, without the necessity of cotransfection with a vector providing expression of RAR.